Method of production of sophorosides by fermentation with fed batch supply of fatty acid esters or oils

ABSTRACT

The invention relates to a fed batch production process for a composition of sophorosides, in which culturing takes place of at least one Candida bombicola or Candida apicola strain and the cultured strain is exposed in a reaction zone to an excess sugar supply and a continuous supply of at least one appropriate substrate at a supply rate to the reaction zone between 0.01 and 4 grams per hour and per liter of initial reaction volume and for a supply time such that the residual concentration of the substrate in the reaction zone is maintained at a value at the most equal to 18 grams per liter of initial reaction volume for the supply time and the composition of sophorosides produced is recovered.

BACKGROUND OF THE INVENTION

The invention relates to a process for the production, with a continuoussupply of the substrate (fed batch) of a sophoroside composition byfermentation. Sophorosides are e.g. used in cosmetology, in dandrufftreatment for hair and as a bacteriostatic agent in deodorants,particularly in lactone form (EP-B-209783.

It is stated in U.S. Pat. Nos. 3,205,150 and 3,312,684 that a quantityof sophorosides was produced by a Fermentation process using aTorulopsis bombicola culture the strain presently classified as Candidabombicola.

Sophorosides are considered as being a mixture of the compoundsrepresented by Formulas (1) and (2): ##STR1## in which R¹ representshydrogen or an acetyl group and R² hydrogen or an alkyl group having 1to 9 carbon atoms, when R³ is a saturated hydrocarbon radical with 7 to16 carbon atoms, or R² represents hydrogen or a methyl group, when R³ isan unsaturated hydrocarbon radical with 13 to 17 carbon atoms.

These compounds can be used as cleaning agents and emulsifiers and haveexcellent hygroscopic and hydrophilic properties due to the sophorosegroup and hydrophobic properties as a result of the Fatty acid.

However, sophorosides are a group of numerous homo logs and theformation ratio of these homologs varies as a function of theirsubstrate, e.g. a hydrocarbon source, as well as the fermentationconditions (FR-A-2399438). Consequently, the properties and functions ofthese compounds vary with the ratios of the homologs, bearing in mindthat use is generally made of a group of said homologs and it hashitherto been difficult to be able to produce a product having a givenratio using a fermentation process.

The prior art is illustrated by the document "Journal of the AmericalOil Chemists Society, vol.65, No. 9, September 1988, Champaign, Ill.,USA, pages 1460-1466: H. J. ASMER et al-- "Microbial production,structure elucidation and bioconversion of sophorose lipids."

Sophorosides are generally prepared in the presence of a substrate, suchas is e.g. described in U.S. Pat. No. 3,205,150. for example, saidsubstrate can be in the form of hydrocarbons, saturated or unsaturatedfatty acids, acid esters including glycerides, vegetable oils such assoybean oil, etc. Supply generally takes place discontinuously at timeintervals of approximately 12 to 24 hours and with a quantity ofapproximately 2% by weight, based on the initial reaction volume, foreach addition.

It is stated that the presence of a higher substrate quantity (3 to 4%approximately) leads to a reduction in the yield of sophorosidesproduced. Moreover, 24 to 48 hours following the final substrateaddition, no supplementary conversion is observed.

This batch production process is also described in U.S. Pat. No.4,297,340, where a substrate quantity (150 g) is added every 24 hoursfor 6 days, the total culture production time being 7 days.

These discontinuous supply processes make it possible to reach finalcrude sophoroside production levels not exceeding 23%, so that yieldsare limited. The reason for this limitation is the lipolytic capacity ofthe microorganism, which transforms the residual esters or oils of thefermentation medium into fatty acids. These fatty acids are notinhibitors to microorganism growth, but they are liable to significantlyaffect the sophoroside production speed, when already present in themedium.

SUMMARY OF THE INVENTION

The object of the invention is to obviate the disadvantages of the priorart. A process for the preparation of sophorosides has been discovered,which in particular makes it possible to improve the productivity of thecultured strain, the yield of sophorosides produced and therefore theproduction of the sought sophorosides.

In a more detailed manner, the invention relates to a process for thefed batch production of a composition of sophorosides, in whichculturing takes place of at least one Candida bombicola or Candidaapicola strain in a culture medium including a nitrogen source underappropriate conditions for producing said strain and the cultured strainis exposed in a reaction zone to a preferably excess sugar supply and acontinuous supply of at least one appropriate substrate under adequateaeration, temperature and pH conditions, characterized in that thefollowing sequence is performed at least once:

a. the strain is supplied with said substrate at a supply flow rate inthe reaction zone between 0.01 and 4 grams per hour and per liter ofinitial reaction volume and for a supply time such that the residualconcentration of said substrate in the reaction zone is kept at a valueat the most equal to 18 grams per liter of initial reaction volume forsaid supply time and

b. the composition of sophorosides produced is recovered. In a preferredmanner, the continuous substrate supply can take place according to atime-decreasing profile.

According to a feature of the process, the sophoroside compositionrecovery stage comprises the stoppage of the substrate supply or theaeration of the reaction zone, the recovery, after settling, of thelower sophoroside phase containing water and at least one washing withwater of the sophorosides at a temperature generally between 10° and 90°C. for e.g. 10 to 60 min.

The strain used is advantageously Candid bombicola CBS 6009. Theproductivity observed is then excellent and the yield of sophorosidesproduced is well above that of the prior art.

The process can be performed according to two embodiments. In the firstembodiment, the substrate is supplied by regulating the supply rate tothe reaction zone under the aforementioned operating conditions, thesubstrate supply being stopped when the total injected substratequantity reaches approximately at the most 280 g·l⁻¹ of initial reactionvolume and the sophoroside composition is recovered in the mannerindicated hereinbefore. According to a second, more advantageousembodiment, the substrate is supplied by regulating the supply rate inthe reaction zone under conditions such that the residual substrateconcentration is close to a preferably fixed value generally between 0.1and 18 and preferably between 0.5 and 3 g·l⁻¹. As soon as the O₂concentration in the dissolved reaction zone advantageously approaches0, the substrate supply and the stirring of the fermenter are stopped,the solution is allowed to settle, the sophoroside phase is recoveredand washed at least once at a temperature between 10° and 90° C. andsubstrate is again supplied to the reaction zone freed from saidsophoroside phase. It is thus possible to recommence this supply,settling and recovery sequence until the microorganism biosynthesiscapacity is exhausted.

According to another feature of the process and the continuous supply,the reaction zone can initially optionally contain a sugar in excess anda substrate concentration of 0.5 to 40 g·l⁻¹ of initial reaction volumeand advantageously 1 to 25 g·l⁻¹ and said strain is continuouslysupplied with substrate after a period of e.g. at the most 48 hours,i.e. when the initial substrate concentration is generally between 0.1and 15 g·l⁻¹ per liter of initial reaction medium and preferably between0.1 and 5 g·l⁻¹.

According to another advantageous feature of the process making itpossible to obtain good results, the continuous substrate supply rate inthe reaction zone can be between 1.0 and 3.0 g·h⁻¹ ·l⁻¹ of initialreaction volume and is preferably between 2.0 and 2.5 g·h⁻¹ ·l⁻¹.

This substrate generally incorporates at least one animal oil, at leastone vegetable oil and/or at least one ester of said oil, said oils andsaid esters incorporating an aliphatic, straight chain in saturated orunsaturated form with 10 to 24 carbon atoms. Among the preferred oilsand esters reference is made to the oil and methyl or ethyl esters ofcolza oil, sunflower seed oil, palm oil or soybean oil. Excellentresults are obtained with said esters.

The culture medium can incorporate a mineral nitrogen source (in theform of ammonium ions) and/or organic nitrogen, e.g. in the form ofamino acids such as in particular yeast extract, soybean peptone, caseinhydrolyzates, maize/corn maceration liqueur, wheat gluten hydrolyzatesand meat extracts. The addition of mineral elements such as e.g.potassium, sodium, magnesium and oligoelements such as iron, manganese,molybdenum in the form of their salts (sulphates, phosphates, chlorides)can also make it possible to further improve growth. The culture mediumcan incorporate at least one sugar such as glucose or saccharose.

The culture conditions are generally temperature 18° to 35° C. and pH 3to 8. A good activity level has been obtained at a temperature between20° and 30° C. in a pH range of 3 to 5 and excellent activity resultsare observed at a temperature between 22° and 28° C. and a pH between3.5 and 4. Fermentation takes place under initial asepsis and aerobiosisconditions.

According to another feature of the process, it is possible to introducethe strain contained in the culture medium into the reaction zone inorder to expose it to the substrate and sugar supply, but according toanother feature of the process the strain can be removed from theculture medium by known procedures and introduced into the reactionzone, where it is exposed to the substrate and sugar supply.

The sophoroside production process is generally performed under thefollowing conditions: temperature 18° to 35° C., pH 2.5 to 8 andadvantageously 3 to 4, aeration flow rate 0.2 to 2 v.v.m. under apressure of 1 to 5 and preferably 1 to 2 bar (1 bar=0.1 MPa).

Throughout the production time, the pH is checked and regulated to adesired value within the range described hereinbefore by e.g. addingpotash or soda solution.

The quantity of cells used compared with the initial reaction volume isgenerally 1 to 100 g of dry weight per liter and preferably 10 to 30 gof dry weight per liter.

The invention will be better understood from the followingnon-limitative, illustrative examples:

EXAMPLE 1

This example describes a fermentation performed according to theinvention. The strain Candida bombicola CBS 6009 is used for seeding amedium which, independently of the glucose, is deprived of substrate,which is continuously added following seeding. It is colza ethyl ester.The following culture medium is used:

    ______________________________________                                        glucose                 100 g · l.sup.-1                             (NH.sub.4).sub.2 SO.sub.4                                                                             4 g · l.sup.-1                               KH.sub.2 PO.sub.4       1 g · l.sup.-1                               MgSO.sub.4, 7H.sub.2 O  0.5 g · l.sup.-1                             dried corn maceration liqueur                                                                         5.0 g · l.sup.-1                             ______________________________________                                    

Glucose and MgSO₄, 7H₂ O are sterilized in a 4 liter capacity laboratoryfermenter dissolved in 1620 ml of water, whereas the KH₂ PO₄, (NH₄)₂ SO₄and corn liqueur are separately sterilized in an Erlenmeyer flaskdissolved in 180 ml of water. The sterilization of the two solutionstakes place in the autoclave at a temperature of 120° C. and for 30minutes and the culture medium is reconstituted after cooling the twosolutions. It is seeded by 200 ml of a preculture prepared in anErlenmeyer flask with 200 ml of the same medium as the fermentationmedium, but to which has been added 1 ml of colza ethyl ester. Thisflask is seeded by approximately 1 g of a congelate of the Candidabombicola strain. It is incubated, accompanied by stirring, at 25° C.and after 24 hours supplies the fermenter preculture. The productionfermentation takes place in the fermenter having a 4 liter capacity andon an initial reaction volume of 2 liters.

The medium is stirred by using a RAYNERl turbine, whose rotation speedis 1000 r.p.m. Aeration is fixed at 0.5 v.v.m. of air under atmosphericpressure. Following auto-acidification of the culture, the pH of themedium is kept at a constant value of 3.5 by a 4N soda solution, whoseaddition to the reactor is controlled by an electrovalve dependent on apH-meter. The content of oxygen dissolved in the culture medium iscontinuously measured by means of a polarographic electrode connected toa recorder.

In order to prevent any glucose medium limitation, said sugar is added 5times in solid form. On the three first occasions at times t=24 h, t=48h, and t=72 h, each corresponding to 45 g of glucose per liter ofinitial medium, whereas the two latter at t=96 h and t=120 h eachcorrespond to 32.5 g of glucose per liter. The colza ethyl ester supplyis provided by means of a peristaltic pump, whose supply speed is fixedat 2 g·l⁻¹ ·h⁻¹ up to times t=96 h and then 1 g·l⁻¹·h⁻¹ up to time t=144h. Sophoroside production is followed by taking samples and measurementtakes place by the method of Gobbert et al (1984, Biotechnol. lett. 6,225-230). This production, which starts with the growth of themicroorganism, increases with colza ethyl ester addition. The residualconcentration of the latter, measured by gas chromatography after hotextraction with heptane, is equal to 0.7 g·l⁻¹.

After 144 h fermentation, stirring, aeration and ethyl ester supply arestopped and the crude sophorosides are allowed to settle for 1 hour.They are recovered and stirred with 1.5 l of distilled water at 45° C.They are again left to settle for 4 hours and a second washing takesplace under the same conditions. Following the second washing, 565 g ofcrude sophorosides per liter of initial second are recovered. Theproductivity of the fermentation, calculated on the basis of the crudesophorosides and the duration of the test, is therefore 3.92 g of crudesophorosides per liter of initial medium and per hour. The water contentof the crude sophorosides measured by the Karl-Fischer method is 45%.Taking account of the sugar arid ester quantities supplied and equalrespectively to 300 and 240 g·l⁻¹, the anhydrous sophoroside productionyield based on the sum of the glucose plus ester is 59.5%. Theproduction, productivity and yields were also measured after 96 hours ofculture (immediately prior to the fourth glucose addition) forcomparison with the discontinuous ester supply protocol (example 2).After 96 hours, the crude sophoroside production corresponds to 295g·l⁻¹ and therefore to a productivity of 3.07 g·l⁻¹. The conversionyield of "glucose plus ethyl ester" in anhydrous sophorosides is then38%.

EXAMPLE 2

(Comparison)

Example 1 is recommenced replacing the continuous colza ethyl estersupply by an addition every 12 hours of 20 g·l⁻¹ of colza ethyl ester.The first addition took place immediately following the seeding of thefermenter. As in example 1, the crude sophoroside production kinetics inthe fermentation medium is followed. It starts at the end of growth, butstops prematurely after 96 hours, at the same time as the 9th colzaethyl ester addition. On continuing ethyl ester additions, it would alsobe impossible to recover by settling the sophorosides, which remainintimately mixed with the residual ethyl ester. After 96 hours only 210g·l⁻¹ of crude sophorosides have been recovered. The productivity, basedon the crude sophorosides and the 96 hour period, is equal to 2.18g·l⁻¹·h⁻¹. The conversion yield of "glucose (235 g·l⁻¹) plus colza ethylester (180 g·l⁻¹)" in anhydrous sophorosides (moisture content 45%) is27.8%. These performance figures are below those obtained for the sametime in example 1.

EXAMPLE 3

Example 1 is repeated up to t=95, at which there is a substantiallytotal exhaustion of the dissolved oxygen of the medium. During thisfirst period, the residual colza ethyl ester concentration in thefermentation medium is 0.5 g·l⁻¹. Stirring, aeration and ester supplyare stopped for 15 minutes and the sophorosides which have settled aredrawn off. Aeration, stirring and ester supply of the fermenter arerecommenced and the test is continued in the following way: 45 g·l⁻¹ ofglucose are added at t=96 h and t=120 h and then 32.5 g·l⁻¹ at t=144 h.At the 96th hour, there is a reduction in the ethyl ester supply ratefrom 2 to 1.6 g·l⁻¹ ·h⁻¹ and the supply is continued at this rate up tot=144 h. At this time, the ester supply rate is again reduced to 0.92g·l⁻¹ ·h⁻¹ to t=168 h. This is followed by a second recovery ofsophoroside by settling and the microorganism biosynthesis capacity hasnot been exhausted. The crude sophorosides recovered at t=95 h and t=168h are combined and washed twice, as in example 1. This gives 705 g·l⁻¹of crude sophorosides after 168 h of culture. The fermentationproductivity based on the duration of the test is 4.19 g·l⁻¹ ·h⁻¹ for asupplied sugar quantity of 357.5 g·l⁻¹ and an ethyl ester quantity of290 g·l⁻¹. The anhydrous sophoroside yield is 59.8%, based on the twosubstrates.

EXAMPLE 4

Example 1 is repeated adding 24 g·l⁻¹ of colza ethyl ester to theculture medium and the continuous ethyl ester addition is started 12hours following seeding. The injection speed is fixed at 2 g·l⁻¹ ·h⁻¹ upto t=96 h and then 1 g·l⁻¹ ·h⁻¹ up to 144 h. After 144 hours, 550 g ofcrude sophorosides are recovered per liter of initial medium. Theproductivity relative to the crude sophorosides is 3.82 g·l⁻¹ ·h⁻¹ andthe anhydrous sophoroside yield is 56%.

EXAMPLE 5

Example 1 is repeated replacing the colza ethyl ester by colza methylester. At the end of the test, 525 g of crude sophorosides arerecovered, which corresponds to a productivity of 3.64 g·l⁻¹ ·h⁻¹ ofcrude sophorosides and a yield of 53.4% of anhydrous sophorosides, basedon the sum "glucose plus ester".

EXAMPLE 6

Example 1 is repeated replacing the colza ethyl ester by sunflower seedmethyl ester. At the end of the Lest 505 g·l⁻¹ of crude sophorosides arerecovered, which corresponds to a productivity of 3.50 g·l⁻¹ ·h⁻¹ and ayield of 51.4% of anhydrous sophorosides, based on the sum "glucose plusmethyl ester".

EXAMPLE 7

Example 1 is repeated replacing the colza methyl ester by colza oil. Theoil injection speed is fixed at 1.2 g·l⁻¹ ·h⁻¹ up to t=96 h and then0.60 g·l⁻¹ ·h⁻¹. The first three glucose additions at t=24 h, t=48 h andt=72 h correspond in each case to 30 g·l⁻¹, while the two last at t=96 hand t=120 h each correspond to 20 g·l⁻¹ of initial medium. After e.g.144 hours culture, there is a recovery of 380 g of crude sophorosidescorresponding to a productivity of 2.64 g·l⁻¹ ·h⁻¹ of crude sophorosidesand a 53% yield of anhydrous sophorosides (moisture content 45%).

We claim:
 1. In a process for the fed batch production of a compositionof sophorosides, comprising culturing a single strain of Candidabombicola or Candida apicola in a culture medium containing a nitrogensource and a substrate under aerated culturing conditions in a reactionzone, the improvement comprising:(a) continuously feeding said substrateto the strain in the reaction zone at a flow rate of between 0.01 and 4grams of substrate per hour and per liter of initial reaction volume,and maintaining the residual concentration of said substrate in thereaction zone at a concentration not exceeding 18 gram per liter ofinitial reaction volume while said substrate is being continuously fedto the reaction zone, (b) recovering the resultant composition ofsophorosides,wherein said substrate is at least one of an animal oil, avegetable oil, or an ester of said animal or vegetable oil, said animalor vegetable oil or said ester comprising an aliphatic, straight chainwith 10 to 24 carbon atoms.
 2. A process according to claim 1, whereinthe strain is Candida bombicola.
 3. A process according to claim 1,wherein the strain is Candida apicola.
 4. A process according to claim1, wherein the sophoroside composition recovery stage (b) comprisesstopping the substrate supply or the aeration of the reaction zone;permitting the resultant culture medium to settle to form a lowersophoroside phase containing water; recovering said lower phase andwashing the lower phase of sophorosides with water at a temperaturebetween 10° and 90° C. for 10 to 60 minutes.
 5. A process according toclaim 1, wherein the substrate supply is stopped when the oxygenconcentration in the reaction zone is substantially equal to
 0. 6. Aprocess according to claim 1, wherein the substrate supply is stoppedwhen the total injected substrate quantity reaches at the most 280 g·l⁻¹of initial reaction volume.
 7. A process according to claim 1, whereinsophoroside production takes place at a temperature of 18° to 35° C., ata pH of 2.5 to 8, in the presence of a sugar excess and in which thereaction zone is aerated at a rate of 0.2 to 2 v.v.m. under a pressureof 1 to 5 bar.
 8. A process according to claim 1, wherein the strain isobtained from an ex-situ prepared culture.
 9. A process according toclaim 1, wherein the cell concentration is 1 g to 100 g of dry weightper liter of reaction volume.
 10. A process according to claim 1,wherein the residual substrate concentration is maintained at between0.5 and 3 g·l⁻¹.
 11. A process according to claim 1, wherein saidcontinuous feeding is initiated directly after the reaction zone isseeded with said strain.
 12. A process according to claim 1, wherein theculture medium comprises sugar.
 13. A process according to claim 12,wherein the sugar is added to the culture medium in an excessconcentration.
 14. A process according to claim 1, wherein the substrateflow rate into the reaction zone is between 1.0 and 3.0 g·l⁻¹ ·h⁻¹ ofinitial reaction volume.
 15. A process according to claim 14, whereinthe flow rate is between2 and 2.5 g·h⁻¹ ·l⁻¹.
 16. A process according toclaim 1, wherein the strain contained in the culture medium is culturedwith the substrate and with added sugar.
 17. A process according toclaim 16, wherein the sugar is added to the culture medium in an excessconcentration.
 18. A process according to claim 16, wherein said flowrate of continuous feeding of the substrate is decreased at least onceduring said culturing and the culturing is continued at said decreasedflow rate of continuous feeding.
 19. A process according to claim 1,wherein the reaction zone initially contains a substrate concentrationof 0.5 to 40 g·l⁻¹ of initial reaction volume, and the substrate iscontinuously supplied to said strain after the initial substrateconcentration is 0.1 to 15 g·l⁻¹.
 20. A process according to claim 19,wherein the substrate is continuously supplied to said strain when theinitial substrate concentration is between 0.1 and 5 g·h⁻¹ ·l⁻¹.
 21. Aprocess according to claim 1, wherein said flow rate of continuousfeeding of the substrate is decreased at least once during saidculturing and the culturing is continued at said decreased flow rate ofcontinuous feeding.
 22. A process according to claim 21, wherein saidflow rate is decreased at least twice and the culturing is continued atsaid decreased flow rates of continuous feeding.
 23. In a process forthe fed batch production of a composition of sophorosides, comprisingculturing a strain of Candida bombicola CBS 6009 in a culture mediumcontaining a nitrogen source and a substrate under aerated culturingconditions in a reaction zone, the improvement comprising:(a)continuously feeding said substrate to the strain in the reaction zoneat a flow rate of between 0.01 and 4 grams of substrate per hour and perliter of initial reaction volume, and maintaining the residualconcentration of said substrate in the reaction zone at a concentrationnot exceeding 18 grams per liter of initial reaction volume while saidsubstrate is being continuously fed to the reaction zone, and (b)recovering the resultant composition of sophorosides,wherein saidsubstrate is at least one of an animal oil, a vegetable oil, or an esterof said animal or vegetable oil, said animal or vegetable oil or saidester comprising an aliphatic, straight chain with 10 to 24 carbonatoms.